The thermal stability of the H3/H4 core histone complex has been studied in low ionic strength conditions by differential scanning calorimetry and circular dichroism spectroscopy. At neutrality, the major molecular species present in solution is the (H3-H4)2 tetramer. Its thermodynamic properties cannot be studied directly though, since its thermal denaturation is irreversible even at the lowest salt concentrations. However, a complete thermodynamic analysis can be performed at low ionic strength and pH 4.5, where the (H3-H4)2 tetramer is quantitatively dissociated into two H3-H4 dimers, and reversibility of the thermal transitions is attained. The unfolding transition temperature of the 26.5 kDa H3-H4 dimer increases as a function of both the ionic strength of the solvent and the total protein concentration. The thermal denaturation of the H3-H4 dimer is characterized by the presence of a single calorimetric peak, centered at 58 ~ C , with a corresponding enthalpy change of 25 kcal/mol of 13 kDa monomer unit, and a change in heat capacity upon unfolding of 0.6 kcal/(K mol of 13 kDa monomer unit). The H3/H4 associative system (tetramer or dimer) is stable between pH 9.5 and 3.0. At pH 1.5, the system is unfolded at all temperatures. At low ionic strength conditions and pH values between 5.0 and 2.5, the H3-H4 dimer behaves as a highly cooperative system, melting as a single unit without any individual H3 and H4 folded monomers detected during the treatment. Just like for the H2A and H2B histones, the H3 and H4 polypeptides are properly folded only when assembled as H3-H4 dimers or in higher order histone complexes. Therefore, coupling along the interfaces of the two chains within the heterodimer is the major factor contributing to the stabilization of the secondary and tertiary structures of the chains, as well as of the histone dimers.